Heterotrimeric G proteins (123) mediate the majority of signaling pathways in mammalian cells. It is long held that G protein function is localized to the plasma membrane. Observation of the spatio-temporal dynamics of G protein localization in living mammalian cells using various imaging techniques showed unexpectedly that G protein subunits shuttle rapidly (t1/2 <1 min) between the plasma membrane and intracellular membranes. Consistent with such shuttling, G protein pools are constitutively maintained in endomembranes and receptor activation results in six out of the twelve members of the G protein 3 subunit family translocating to endomembranes as 23 complexes. The long term goal is to determine if shuttling and receptor mediated translocation are widespread among various eukaryotes and are central processes in signaling that have remained undiscovered. The specific aims are (i) to identify mechanisms at the basis of G protein shuttling, 23 translocation and differential targeting to intracellular membranes. (ii) To test the hypothesis that receptor induced 23 translocation controls the magnitude of amplification of downstream signaling. (iii) To identify the role of the translocated 23 complex in endomembranes. Sensitive methods that measure receptor-G protein interaction will be used to test the model that affinity of 3 subunit types for receptors regulates translocation. The finding that shuttling and translocation are acylation dependent and a palmitoylated PDZ domain protein inhibits translocation will be followed up to identify potential binding partners that mediate shuttling through an acylation/deacylation cycle. Live cell measurements of G protein effector activity will be used to examine the role of receptor induced 23 complex translocation on downstream activity at the plasma membrane. The possibility that translocation of 23 complexes to endomembranes regulates Ras activity and membrane trafficking will be tested using sensors that report these activities in live cells. The unexpected finding that G proteins continually shuttle between the PM and endomembranes facilitating receptor mediated translocation of a large number of 23 subunit types indicates the presence of novel mechanisms and functions that have not been anticipated. Elucidating these mechanisms and functions can provide newer therapeutic sites and targets for diseases caused by aberrant signaling. More than 30% of the commercially available therapeutic drugs today are targeted at G protein coupled receptors. The rapid reversible translocation of fluorescent protein tagged 3 subunits in response to agonists and antagonists provides a tool for non-invasively and rapidly performing high throughput and high content screening for potential therapeutic molecules that act on these receptors.G proteins mediate most signaling pathways in cells and their receptors are one of the most important targets of therapeutic drugs. Unanticipated movement of G proteins between the external and internal membranes of cells has been discovered. Identifying the mechanistic and functional bases of this movement can provide newer therapeutic sites and targets for diseases caused by aberrant signaling.